Center for Veterinary Biologics SAM 513.11 

Testing Protocol Page 1 of 14

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 513

Supplemental Assay Method for the Determination of Protein and Phenol in 

PPD (Purified Protein Derivative Produced from Cultures of Mycobacterium 

bovis Strain AN-5) Bovis Tuberculin

Date: June 17, 2022 

Number: SAM 513.11 

Supersedes: SAM 513.10, February 8, 2016 

Standard Requirement: 9 CFR 113.409 

Contact: Email: Methodsrequest.notification@usda.gov 

Phone: Center for Veterinary Biologics, 515-337-6100 

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844 

Ames, IA 50010 

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.

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Testing Protocol Page 2 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

Table of Contents

1. Introduction

2. Materials

2.1 Equipment

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents 

3.4 Preparation of the sample 

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

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Testing Protocol Page 3 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

1. Introduction

This Supplemental Assay Method (SAM) describes the procedures for determination of protein 

concentration and phenol content in Purified Protein Derivative (PPD) produced from cultures of 

Mycobacterium bovis strain AN-5; as prescribed in title 9, Code of Federal Regulations, 

(9 CFR), part 113.409. 

Protein concentration is determined by the Microkjeldahl test for nitrogen. Phenol is determined 

by direct titration with a standardized bromide-bromate solution. 

2. Materials

2.1 Equipment

Equivalent equipment may be substituted for any brand name listed below.

2.1.1 Balance, top loading, capable of measuring 0.001 g 

2.1.2 Digestion unit (Büchi) 

2.1.3 Distillation unit (Büchi) 

2.1.4 Volumetric pipettes, Class A, 5-, 10-, and 25-mL 

2.1.5 Volumetric flasks with barrel head glass stopper, Class A, 500-mL and 

1-L

2.1.6 Erlenmeyer flasks, 125-mL 

2.1.7 Burets with PTFE stopcocks, precision bore, Class A, 10-, 25-, and 50-mL 

2.1.8 Weigh boats, or equivalent 

2.1.9 Graduated cylinders, Class A, 50-, 100-, 250-, 500-mL, and 1-L 

2.1.10 Glass-stoppered Erlenmeyer flasks, 250-mL 

2.1.11 Heating/stirring plate with stirring bars

2.1.12 Filter paper, 11μm particle retention (Whatman No. 1) 

2.1.13 Timers, 30 seconds to 2 minutes 

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Testing Protocol Page 4 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

2.1.14 Dropper, i.e., transfer pipette, Pasteur pipette, dropper bottle 

2.1.15 Pipettor and tips to accurately dispense 100- to 1000-μL 

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

chemicals are reagent grade, unless specified. 

2.2.1 Protein test

1. Sulfuric acid (H2SO4), CAS# 7664-93-9, Purity: Minimum 95.0%,

Maximum 98.0%

2. Kjeldahl Catalyst Tablets, 1.5 g K2SO4 + 0.075 g HgO

3. Sodium hydroxide (NaOH), CAS# 1310-73-2, Purity: 98.5%

4. Boric acid (H3BO3), CAS# 10043-35-3, Purity: 99.9%

5. Methyl red, CAS# 493-52-7, Purity: 98.0%

6. Hydrochloric acid (HCl), CAS# 7647-01-0, Assay: 36.5-38.0%

7. Sodium carbonate (Na2CO3), CAS# 497-19-8, Purity: 99.9%

8. Bromo phenol blue, CAS# 115-39-9, Purity: 98.0%

9. Control Sample – Either a pool of PPD tuberculin products with

established protein and phenol values as tested by PL-CAS; or a product

produced for use as a control sample and tested by PL-CAS.

10. Protein Standard Reference Material, Bovine Serum Albumin (current

lot of SRM 927 from National Institute of Standards and Technology)

2.2.2 Phenol test (some reagents same as for protein) 

1. Hydrochloric acid (HCl), CAS# 7647-01-0, Assay: 36.5-38.0%

2. Water (H2O), Purity: distilled, demineralized, reverse osmosis or

equivalent.

3. Methyl orange, CAS# 547-58-0, Purity: 98.0%

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Testing Protocol Page 5 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

4. Silicotungstic acid hydrate (H4[Si(W3O10)4]*26H2O), CAS# 12027-43-

9, Purity: 99.0%. Store at 4°C.

5. Sulfuric acid (H2SO4), CAS# 7664-93-9, Purity: Minimum 95.0%,

Maximum 98.0%

6. Arsenic trioxide, anhydrous (As2O3), CAS# 1327-53-3, Purity: 99.9%

7. Sodium hydroxide (NaOH), CAS# 1310-73-2, Purity: 98.5%

8. Phenol (C6H6O), CAS# 108-95-2, Purity: ≥ 99.0%

This can be a purchased NIST standard and diluted, if necessary, to the

appropriate level.

9. Sodium bicarbonate (NaHCO3), CAS# 144-55-8, Purity: 99.9%

10. Potassium bromate (KBrO3), CAS# 7758-01-2, Purity: 98.5%

11. Potassium bromide (KBr), CAS# 7758-02-3, Purity: 99.0%

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel must have experience or training in this protocol. This includes

working knowledge of the use of general laboratory equipment, glassware and chemical

safety, and specific training in the operation of the laboratory equipment and reagents

listed in Section 2.

Analysts performing this procedure should first conduct at least 2 trial runs using controls

and standards and obtain results within acceptable limits.

3.2 Preparation of equipment/instrumentation

All equipment must be operated according to manufacturers’ recommendations and

monitored in compliance with applicable standard operating procedures.

3.2.1 Protein Test

1. Prepare digestion and distillation units according to manufacturer’s

recommendations.

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Testing Protocol Page 6 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

2. Check levels of water and sodium hydroxide tanks on the distillation

unit, fill if necessary.

3. Prime the buret by rinsing with standardized HCl.

3.2.2 Phenol Test

Prime the buret by rinsing with test fluid. 

3.3 Preparation of reagents 

Reagents are stable for 6 months from date of preparation and stored at room 

temperature, unless otherwise noted. Prepare reagents in volumes appropriate to demand 

to minimize waste due to expiration. 

Glassware used for preparation of reagents must meet ASTM requirements; 

measurements are based on the measurements of uncertainty outlined in those 

requirements.

All references to “water” indicate distilled, demineralized, reverse osmosis, or water of 

equivalent purity (Section 2.2.2.2). 

In the following steps the acronym QS is used. It is defined as quantity sufficient; as 

much as is sufficient.

3.3.1 Protein test (all reagents stable for at least 6 months unless specified)

1. Standard, 1.0 ± 0.1 mg/mL Protein: Dilute protein standard reference

material (Section 2.2.1(10)) to the range of 1.0 ± 0.1 mg/mL protein with

water. Prepare sufficient dilution to provide several 15-mL portions. Store

at 4°C.

2. Control Sample: Either (1) a pool of PPD tuberculin products with

established protein and phenol values as tested by PL-CAS; or (2) a

product produced for use as a control sample and tested by PL-CAS.

(1) Combine any sample volumes remaining after test completion in a

pool. Record all identifying information, CAS protein result and

expiration date for each sample. Control sample protein concentration is

the mean of CAS results for all samples included in the pool.

(2) Obtain a product produced for use as a control sample. Analyze the

product a total of three times, mean of these trials must be 1.0 ± 0.1

mg/mL protein. Expiration date is as indicated on product. Store at 4° ±

10° C.

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Testing Protocol Page 7 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

3. 32% Sodium Hydroxide (NaOH): Caution!! NaOH is caustic--

Avoid contact with skin. Dissolve 640 g ± 1 g sodium hydroxide in

approximately 1.4 L water in a 2-L volumetric flask on a stir plate.

Solution will be HOT!! Cool to room temperature. QS with water.

Store at room temperature.

4. Saturated Boric Acid (H3BO3): Use a container with at least twice as

much volumetric capacity as your final volume. Add 15.0 ± 0.1 g boric

acid to 100 mL water. Stir, with heat, until all boric acid dissolves. Some

boric acid recrystallizes when cool. Store at room temperature.

5. 0.1% bromo phenol blue: Dissolve 0.1 ± 0.1 g in 100 mL water. Store

at room temperature.

6. 0.5% methyl red: Dissolve 0.5 ± 0.1 g in 100 mL ethanol. Store at

4°C.

7. Standardized 0.01 - 0.02N Hydrochloric acid (HCl): Caution!!

Concentrated HCl is corrosive – Handle in fume hood. Avoid contact

with skin. May be prepared and standardized or a standardized solution

may be purchased.

Preparation: Add 1.72 ml hydrochloric acid to approximately 900 mL 

water in a 1-L volumetric flask. QS with water. Store at room temperature. 

Standardization: Weigh approximately 0.010 g dried sodium carbonate. 

Record weight. Dissolve in 25 mL water. Add three drops 0.1% bromo 

phenol blue (indicator). Titrate with prepared 0.01 – 0.02N hydrochloric 

acid to an endpoint color of green, not bluish green nor yellowish green. 

Calculate the normality of the hydrochloric acid solution as below. 

Perform three trials and use the calculated mean as the normality of the 

hydrochloric acid solution. 

Calculation: 

NN HHHHHH = 

[(gg NNNN2 CC3)(1000)]

[(mm HHHHHH)(52.994)]

3.3.2 Phenol test 

1. Standard, 0.50% phenol: Caution!! Handle in fume hood. Avoid

contact with skin. Dissolve 5.0 ± 0.01g phenol in approximately 500 mL

water in a 1-L volumetric flask; QS to 1L with water.

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Testing Protocol Page 8 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

2. Control Sample: Either (1) a pool of PPD tuberculin products with

established protein and phenol values as tested by PL-CAS; or (2) a

product produced for use as a control sample and tested by PL-CAS.

(1) Combine any sample volumes remaining after test completion in a

pool. Record all identifying information, CAS phenol result, and

expiration date for each sample. Control sample phenol concentration

is the mean of CAS results for all samples included in the pool.

(2) Obtain a product produced for use as a control sample. Analyze the

product a total of three times, mean of these trials must be 0.50 ± 0.04

% phenol. Expiration date is as indicated on product. Store at

4°± 10° C.

3. 20% Hydrochloric Acid (HCl): In a 1-L volumetric flask, slowly add

200 mL hydrochloric acid to 600 mL water; QS to 1 L with water.

4. 0.1% Methyl Orange: Dissolve 0.1 ± 0.01 g methyl orange in

100 mL water. Filter if necessary.

5. Silicotungstic acid solution (SAS): Dissolve 60.00 ± 0.5 g

silicotungstic acid hydrate in 400 mL water in a 500-mL volumetric flask.

Add 50 mL sulfuric acid. When cool, QS to 500 mL with water.

6. Clarifying solution (CS): Add 50 mL SAS and 125 mL 20%

hydrochloric acid to 325 mL water. Prepare fresh prior to each test.

7. "Acid solution" for As2O3 standardization solution: Add 110 mL

hydrochloric acid and 2.5 mL 0.1% methyl orange to 100 mL water.

8. 0.050 N Arsenic trioxide (As2O3): CAUTION!! Arsenic trioxide is

extremely toxic. Avoid contact; handle in fume hood using gloves, mask,

and goggles. Consult the Safety Data Sheet for specific handling

instructions before proceeding. Dissolve 2.4730 ± 0.001 g anhydrous

arsenic trioxide in 25 mL hot 1 N sodium hydroxide in a 1-L volumetric

flask. Neutralize solution with 25 mL 1 N sulfuric acid. When cool, QS to

1 L with water.

9. 1N Sodium hydroxide: Dissolve 4.00 ± 0.01 g of sodium hydroxide in

60 mL water in a 100-mL volumetric flask; QS to 100 mL with water.

10. 1N Sulfuric acid: In a 100-mL volumetric flask, slowly add

4.904 mL sulfuric acid to 60 mL water; QS to 100 mL with water.

11. Test fluid (TF): Dissolve 0.30 ± 0.01 g sodium bicarbonate, 1.67 ±

0.01 g potassium bromate, and 15.00 ± 0.01 g potassium bromide in water

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Testing Protocol Page 9 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

and QS to 1 L with water. CRITICAL CONTROL POINT: The test fluid 

must be standardized as described in Section 3.3.11(1) prior to use. 

1. Standardization

a. Prepare standardization solution: Add 25 mL 0.050N

arsenic trioxide to 10 mL “Acid Solution.”

b. Confirm standardization solution by titrating with previous

lot of TF. It should take 21.3 mL TF to titrate the

standardization solution.

c. Titrate standardization solution with new lot of TF. The

required titration volume is 21.3 mL TF. A first time titration

may require less than 21.3 mL TF, in which case the TF

volume must be adjusted by adding the correct volume of water

to the TF, continue to Step 1d if this is the case. If the first

time titration is 21.3 ± 0.1 mL TF, continue to Step 2.

d. Adjust the TF volume. For this step the calculations are

shown and an example is used to illustrate.

A = Starting volume of TF (mL) 

B = Titration volume of TF (mL) 

C = Volume of TF left (mL) 

D = Required titration volume (21.3 mL) 

E = Adjusted volume of TF (mL) 

F = Volume of water to be added to volume of TF left to 

achieve the adjusted volume (mL) 

Example: Assume the starting volume of TF is 1000 mL and 

the titration volume is 20.5 mL. 

• A – B = C

Example: (1000 mL) - (20.5 mL) = 979.5 mL

• (C) (D)

(B) = E

Example: 

(979.5 mL) (21.3 mL)

(20.5 mL) = 1017.7 mL

• E – C = F

Example: (1017.7 mL) - (979.5 mL) = 38.2mL

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Testing Protocol Page 10 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

• Add the calculated volume of water (F) to the existing TF

and put any TF remaining in the buret back into flask.

Continue to Step 2.

2. Repeat Step 1c until three consecutive trials produce an

average titration volume of 21.3 mL.

3.4 Preparation of the sample

3.4.1 Receipt

Complete sample receipt as described by standard operating procedures. 

3.4.2 Preparation

Licensed or prelicense biologics products are generally received in sealed serum 

bottles and stored at 4°± 10°C prior to testing. Before testing, allow sample vials 

and reagents to come to room temperature. 

4. Performance of the Test

4.1 Protein

Analyze the control pool, protein standard and a reagent blank sample in duplicate each

time testing is performed. Analyze each sample in triplicate.

4.1.1 Place one Kjeldahl Catalyst tablet, 5.0 mL sample and 3.0 mL sulfuric 

acid into a digestion flask

Caution: HgO is poisonous – Use gloves, mask, and goggles. 

Caution: Concentrated H2SO4 is corrosive – Avoid contact with skin.

4.1.2 Place the digestion flasks in the digestion unit. 

4.1.3 Digest in a method that results in a fully digested product, i.e., 250°C for 

15 minutes, 410°C for 60 minutes, and 500°C for 15 minutes. Final product 

should be clear to white-cloudy. 

4.1.4 Cool in digestion flasks, add 6 mL water, mix (a vortex may be used), and 

cool again. 

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Testing Protocol Page 11 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

4.1.5 Place digestion flask and a 125-mL Erlenmeyer flask containing 5 mL 

saturated boric acid solution and 3 drops 0.5% methyl red into the distillation unit. 

Tilt the Erlenmeyer flask so the tip of the condenser is immersed in the boric acid. 

4.1.6 Add a sufficient amount of 32% sodium hydroxide to make the solution in 

the digestion flask alkaline, i.e., 25 mL. A sodium hydroxide pump may be used 

for this, if the distillation unit is equipped with one. 

4.1.7 Distill for two minutes.

4.1.8 Titrate collected distillate to endpoint color change of yellow to deep rose 

(pH 5.0) with standardized hydrochloric acid. Record the volume of hydrochloric 

acid titrated. 

4.2 Phenol

Analyze the control pool and phenol standard in duplicate each time testing is performed. 

Analyze samples in triplicate.

4.2.1 Combine 5 mL sample/standard/control and 100 mL clarifying solution 

(CS) to a 250-mL glass-stoppered flask. Shake 2 minutes. Filter through filter 

paper and collect 50 mL of filtrate. 

4.2.2 Transfer 50 mL of filtrate to another 250-mL glass-stoppered flask. Add a 

stir bar and place flask on stir plate with buret directly above. Add 1 drop 0.1% 

methyl orange (indicator), stir for a few seconds. Observe the color as pink. 

An acceptable alternative to using a stir plate and stir bar would be shaking the 

flask. 

4.2.3 Titrate with 2 mL test fluid (TF), stir or shake for a few seconds. Observe 

the color; if pink, repeat. If colorless, go to Section 4.2.4. 

4.2.4 Stir or shake 30 seconds. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If colorless for ≤ 10 seconds or if pink, titrate with 1 mL TF, 

and repeat. If colorless for ≥ 10 seconds, go to Section 4.2.5. 

4.2.5 Stir or shake 1 minute. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If pink for ≥ 10 seconds, titrate with 0.50 mL TF, and repeat. 

When colorless within 10 seconds, record total volume of TF as the endpoint of 

titration and use this volume for calculation of percent phenol. 

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Testing Protocol Page 12 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

5. Interpretation of the Test Results

5.1 Protein (Report mean of triplicates)

mm PPPPPPPPPPPPPP⁄mmmm = (mmmm ss ss − mmmm bb bb)(NN HHHHHH)(1.4007)(6.25)(10)

5.0 mmmm

mL sample = Volume of standardized HCl required for sample 

mL blank = Volume of standardized HCl required for blank

N HCL = Normality of HCl 

1.4007 = Milliequivalent weight of nitrogen x 100 

6.25 = % Nitrogen to % Protein conversion factor 

10 = Conversion factor for percent protein to mg protein/mL: %P(10)=mgP/mL 

5.0 mL = Volume of sample 

Satisfactory Protein Content*: 1.0 mg/mL ± 0.1 mg/mL 

*This value is to be used unless otherwise noted in the approved Outline of Production

for the product or 9 CFR 113.409.

5.2 Phenol (Report average of triplicates)

Percent phenol = (vol of test fluid) x (0.04) 

Satisfactory Phenol Content*: 0.50% ± 0.04% 

*This value is to be used unless otherwise noted in the approved Outline of Production

for the product or 9 CFR 113.409.

5.3 Controls

Results for controls and standards must be within acceptable limits; otherwise repeat 

testing.

6. Report of Test Results

Report results of the test(s) as described by standard operating procedures.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.409, U.S. Government Printing

Office, Washington, DC.

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Testing Protocol Page 13 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

7.2 AOAC Official Method 960.52, Official Methods of Analysis of AOAC 

International, Arlington, Virginia, 16th Edition, Pat Cuniff, Editor (1995), Volume I, 

Chapter 12, page 7. 

7.3 ASTM Standard E969, Standard Specification for Glass Volumetric (Transfer) 

Pipets. 

7.4 ASTM Standard E288, Standard Specification for Laboratory Glass Volumetric 

Flasks. 

7.5 ASTM Standard E694, Standard Specification for Laboratory Glass Volumetric 

Apparatus. 

8. Summary of Revisions

Version .11 

Updated the coversheet.

Version .10 

• Includes option to purchase a NIST standard phenol.

Version .09

• The document has been revised to provide additional detail.

• 3.3.1: Standardized 0.01 – 0.02 N Hydrochloric acid may be purchased.

• 3.3.1 and 3.3.2: Includes an option for obtaining a product produced for use as a

control sample.

Version .08

• The document has been revised to reflect changes in instrumentation, personnel, and

to provide additional detail.

• 5.1: Protein, the calculation has been changed to reflect that used in AOAC 960.52

taking into account the blank sample and conversion from percent protein to mg

protein/mL. The satisfactory protein content level did not change.

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Testing Protocol Page 14 of 14

Supplemental Assay Method for the Determination of Protein and Phenol in PPD (Purified Protein 

Derivative Produced from Cultures of Mycobacterium bovis Strain AN-5) Bovis Tuberculin

• 5.2: Phenol, the calculation has been changed to reflect the 9 CFR, Part 113.409.

Percent phenol = (vol of test fluid) (0.04). The satisfactory phenol content level did

not change.

• A protein blank sample requirement has been added to the procedure to reflect AOAC

Official Method 960.52.

Version .07

• The document number has been changed from TCSAM513 to SAM 513.

Version .06 

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the name of the contact person has changed. 

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